1. Field of the Invention
This invention relates to improved solid supports for use in heterogeneous fluorescence assays employing a ligand and a specific binding partner to the ligand and, in particular, to improved solid supports for use in fluorescence immunoassays (FIA).
2. Description of the Prior Art
One of the major advances in the clinical laboratory over the past 25 years has been the employment of antibodies to measure extremely small quantities of analytes in biological samples. These immunoassays are characterized by specificities and sensitivities which were unachievable with prior techniques.
The first immunoassay was described in 1959 by Berson and Yalow (Berson, S.A. and Yalow, R.S., Quantitative aspects of the reaction between insulin and insulin binding antibody. J. CLIN INVEST. 38:1996-2016, 1959) and utilized radioactive insulin as a "tracer" probe in a competitive antigen-antibody immunologic reaction. A variety of permutations as to how immunoassays are practiced including the use of radiolabeled antibodies in an immunometric (IRMA) format rather than radiolabeled antigen in a competitive binding format have developed since Berson's and Yalow's initial work.
The use of radiolabeled substances in immunoassays has limited the sites in the clinical laboratory where these assays could be performed due to the biological hazards associated with radioactivity. Also, the need to properly dispose of the radioactive waste materials resulting from these assays has increased their cost. Further, radioisotopes have limited life times as assay reagents due to isotopic decay. As a consequence, there have been continuing efforts to find non-radioactive "tracers" to substitute for radioisotopes in immunoassays. Unfortunately, it has been difficult to develop non-radioactive immunoassays which match the high sensitivity and specificity achieved with radioimmunoassays.
One type of non-radioactive tracer which has been employed in immunoassays with some success and with which the present invention is concerned, is the fluorescent tracer. These tracers have been used in immunoassays since the early 1960's. Reviews of the progress made to date with these tracers can be found in, for example, Cobb, M. and Gotcher, S., Fluorescence immunoassay in the clinical laboratory. AMERICAN J. MED. TECH. 48(8) 671-677, 1982.; Hemmila, I., Fluoroimmunoassays and immunofluorometric assays. CLIN. CHEM. 31(3): 359-370, 1985.; and Nakamura, R.M., Advances in analytical fluorescence immunoassays--methods and clinical applications. In: Nakamura, R.(ed.), CURRENT TRENDS IN CLINICAL LABORATORY ASSAYS, Scripps Research Foundation, La Jolla, Calif., pp. 33-59, 1982.
A major problem with fluorescence immunoassays has been interference due to endogenous serum components. Depending on the particular tracer used, this interference can take the form of absorption ("quenching") of the tracer's output or high background at the wavelength being detected. Depending on the excitation and emission wavelengths of the tracer, serum constituents that may interfere include: proteins, albumin-bilirubin complexes, hemoglobin and vitamins.
The result of this interference by serum components is a loss in assay sensitivity. This loss in sensitivity has tended to limit the application of fluorescence immunoassays to the detection of compounds present in relatively high concentration in the blood, such as, immunoglobulins, the proteins making up complement (C reactive protein) and drugs.
The interference caused by serum components can be reduced or eliminated by removing the interfering components either before or after the immunologic reaction is run. If removed or diluted out before the immunologic reaction takes place, the assay may be run in a "homogeneous" mode, wherein the output of the system is obtained without separating bound and unbound tracer. In general, fluorescence immunoassays employing a homogeneous format have tended to have low sensitivities as a result of the high sample dilutations needed to reduce background fluorescence to acceptable levels.
Heterogeneous formats have also been employed in fluorescence immunoassays. In this mode, the interfering substances are removed after the immunologic reaction takes place by separating the antigen-antibody complex from the reaction mixture. This format generally involves washing the antigen-antibody complex and then resuspending it in solution prior to obtaining an output reading. In order to be able to perform the separation, washing and measurement steps conveniently, the antigen-antibody complex is preferably formed on a solid support. The present invention is directed to providing improved solid supports for use in heterogeneous fluorescence immunoassays, as well as in other types of heterogeneous fluorescence assays in which the presence or concentration of a ligand is determined through the use of a specific binding partner for the ligand.
Prior to the present invention, various solid supports have been employed in heterogeneous fluorescence immunoassays. For example, U.S. Pat. Nos. 4,201,763 (1980) and 4,295,199 (1981), assigned to Bio-rad Laboratories, Inc., Richmond, Calif., describe the use of 0.1-10 micron hydrophilic polymer particles, in particular, 5 micron polyacrylamide beads, to perform heterogeneous fluorescence immunoassays. In this system, after washing, the solid phase antigen-antibody complex is reconstituted into suspension and read in a conventional 90 degree fluorometer. In practice, the Bio-Rad system has been found not to have the required sensitivity to measure small concentrations of analytes and has been restricted primarily to the measurement of immunoglobulins in high concentrations.
Heterogeneous fluorescence immunoassays have also been performed by immobilizing antibodies on flat polymeric surfaces, e.g., surfaces made of polymethylmethacrylate or cellulose nitrate. See Tsay Y.G., Wilson, L., Keefe, E., Quantitation of serum gentamicin concentration by solid phase immunofluorescence method. CLIN.CHEM. 26:1610-1612, 1980. This system has been sold commercially by International Diagnostics, Santa Clara, Calif., under the trade name FIAX-StiQ.
Unlike the Bio-Rad system described above, the FIAX-StiQ system uses front face (zero degree) epifluorometry, rather than conventional 90 degree fluorometry. Detecting the fluorescent output of the probe along the same axis used to excite the probe can produce increased optical sensitivities, as demonstrated below in connection with the preferred embodiments of the present invention. In the FIAX-StiQ system, however, because of its use of a flat surface, full advantage of the front face orientation has not been achieved. Specifically, the FIAX-StiQ system produces a relatively limited utilizable signal due to the low surface area:volume ratio inherent to a flat surface. Only the portion of the flat surface illuminated by the incident beam can produce a fluorescent output signal. Since only a limited number of "tracer" probes can be in the illuminated area due to its limited surface area, the obtained signals are small and lack sensitivity to measure low analyte concentrations. In practice, the FIAX-StiQ system has been used to measure drugs and patient analytes present in high concentration.
Magnetic solid supports have also been used in heterogeneous fluorescence immunoassays. Thus, Pourfarzaneh et al., in an article entitled "Cortisol directly determined in serum by fluoroimmunoassay with magnetizable solid phase," CLIN. CHEM. 26:730-733 (1980), describe the use of magnetizable solid phase cellulose-iron oxide particles to which antibody has been coupled in a fluorescence immunoassay. Unfortunately, these particles are black body absorbers capable of absorbing all the exciting and fluorescent light employed in conventional fluorometry. As a consequence. Pourfarzaneh et al. were forced to add an additional elution step, wherein the fluorescent probe was eluted from the antibody prior to the fluorescence measurement, in order to make their system work.
In addition to the foregoing, Pandex Laboratories, Inc., Mundelein, Illinois, has commercialized a heterogeneous fluorescence immunoassay which it has called "particle concentration fluorescence immunoassay" (PCFIA). See European Patent Publication No. 124,050 (1984) and Jolley et al., Particle Concentration Fluorescence Immunoassay (PCFIA): A New Rapid Immunoassay Technique with High Sensitivity. JOURNAL OF IMMUNOLOGICAL METHODS, 67:21-35 (1984).
This system utilizes front face epifluorescence and polystyrene latex particles having a diameter of 10 microns or less to which antigen or antibody has been chemically coupled. Particles of these sizes will generally stay in suspension for the duration of the immunologic reaction. After the immunologic reaction has been completed, the particles are washed and concentrated by vacuum microfiltration through a membrane filter, e.g., a cellulose acetate or other polymeric filter. Front face epifluorometry is then performed on the concentrated particles. Background due to stray light and light scattered from particles and endogenous fluorescence due to the filter membranes is corrected for each sample by inclusion of a small amount of reference particles sampled at different wavelengths then the "tracer" fluorophore.
According to Pandex, concentrating the particles before measuring their fluorescence results in an increase in sensitivity by increasing the number of "tracer" fluorophores per unit area available for excitation by the incident light beam. As demonstrated below, by using the solid supports of the present invention, such concentration of the solid phase to achieve enhanced sensitivities is not necessary.